The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

Abstract

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.